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1.
Neuroscience ; 465: 128-141, 2021 06 15.
Article in English | MEDLINE | ID: mdl-33951504

ABSTRACT

The Small Optic Lobe (SOL) family of calpains are intracellular cysteine proteases that are expressed in the nervous system and play an important role in neuronal development in both Drosophila, where loss of this calpain leads to the eponymous small optic lobes, and in mouse and human, where loss of this calpain leads to eye anomalies. Some human individuals with biallelic variants in CAPN15 also have developmental delay and autism. However, neither the specific effect of the loss of the Capn15 protein on brain development nor the brain regions where this calpain is expressed in the adult is known. Here we show using small animal MRI that mice with the complete loss of Capn15 have smaller brains overall with larger decreases in the thalamus and subregions of the hippocampus. These losses are not seen in Capn15 conditional knockout (KO) mice where Capn15 is knocked out only in excitatory neurons in the adult. Based on ß-galactosidase expression in an insert strain where lacZ is expressed under the control of the Capn15 promoter, we show that Capn15 is expressed in adult mice, particularly in neurons involved in plasticity such as the hippocampus, lateral amygdala and Purkinje neurons, and partially in other non-characterized cell types. The regions of the brain in the adult where Capn15 is expressed do not correspond well to the regions of the brain most affected by the complete knockout suggesting distinct roles of Capn15 in brain development and adult brain function.


Subject(s)
Calpain , Neurons , Animals , Brain/diagnostic imaging , Brain/metabolism , Calpain/genetics , Calpain/metabolism , Magnetic Resonance Imaging , Mice , Mice, Knockout , Neurons/metabolism
2.
Hum Mol Genet ; 29(18): 3054-3063, 2020 11 04.
Article in English | MEDLINE | ID: mdl-32885237

ABSTRACT

Microphthalmia, coloboma and cataract are part of a spectrum of developmental eye disorders in humans affecting ~12 per 100 000 live births. Currently, variants in over 100 genes are known to underlie these conditions. However, at least 40% of affected individuals remain without a clinical genetic diagnosis, suggesting variants in additional genes may be responsible. Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, an important class of developmental proteins, as yet uncharacterized in vertebrates. We identified five individuals with microphthalmia and/or coloboma from four independent families carrying homozygous or compound heterozygous predicted damaging variants in CAPN15. Several individuals had additional phenotypes including growth deficits, developmental delay and hearing loss. We generated Capn15 knockout mice that exhibited similar severe developmental eye defects, including anophthalmia, microphthalmia and cataract, and diminished growth. We demonstrate widespread Capn15 expression throughout the brain and central nervous system, strongest during early development, and decreasing postnatally. Together, these findings demonstrate a critical role of CAPN15 in vertebrate developmental eye disorders, and may signify a new developmental pathway.


Subject(s)
Calpain/genetics , Eye Abnormalities/genetics , Genetic Predisposition to Disease , Nervous System Malformations/genetics , Animals , Deafness/genetics , Deafness/pathology , Eye Abnormalities/pathology , Female , Humans , Male , Mice, Knockout , Nervous System Malformations/pathology , Neurodevelopmental Disorders/genetics , Neurodevelopmental Disorders/pathology , Pedigree , Phenotype
3.
J Neurochem ; 150(4): 366-384, 2019 08.
Article in English | MEDLINE | ID: mdl-31254393

ABSTRACT

There is considerable evidence from both vertebrates and invertebrates that persistently active protein kinases maintain changes in synaptic strength that underlie memory. In the hermaphrodite marine mollusk, Aplysia californica, truncated forms of protein kinase C (PKC) termed protein kinase Ms have been implicated in both intermediate- and long-term facilitation, an increase in synaptic strength between sensory neurons and motor neurons thought to underlie behavioural sensitization in the animal. However, few substrates have been identified as candidates that could mediate this increase in synaptic strength. PKMs have been proposed to maintain synaptic strength through preventing endocytosis of AMPA receptors. Numb is a conserved regulator of endocytosis that is modulated by phosphorylation. We have identified and cloned Aplysia Numb (ApNumb). ApNumb contains three conserved PKC phosphorylation sites and PKMs generated from classical and atypical Aplysia PKCs can phosphorylate ApNumb in vitro and in cells. Over-expression of ApNumb that lacks the conserved PKC phosphorylation sites blocks increases in surface levels of a pHluorin-tagged Aplysia glutamate receptor measured using live imaging after intermediate- or long-term facilitation. Over-expression of this form of ApNumb did not block increases in synaptic strength seen during intermediate-term facilitation, but did block increases in synaptic strength seen during long-term facilitation. There was no effect of over-expression of this form of ApNumb on other putative Numb targets as measured using increases in calcium downstream of neurotrophins or agonists of metabotropic glutamate receptors. These results suggest that in Aplysia neurons, Numb specifically regulates AMPA receptor trafficking and is an attractive candidate for a target of PKMs in long-term maintenance of synaptic strength. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.


Subject(s)
Membrane Proteins/metabolism , Neuronal Plasticity/physiology , Neurons/metabolism , Protein Kinase C/metabolism , Receptors, AMPA/metabolism , Animals , Aplysia , Protein Transport/physiology
4.
J Neurochem ; 146(4): 429-445, 2018 08.
Article in English | MEDLINE | ID: mdl-29808476

ABSTRACT

The small optic lobes (SOL) calpain is a highly conserved member of the calpain family expressed in the nervous system. A dominant negative form of the SOL calpain inhibited consolidation of one form of synaptic plasticity, non-associative facilitation, in sensory-motor neuronal cultures in Aplysia, presumably by inhibiting cleavage of protein kinase Cs (PKCs) into constitutively active protein kinase Ms (PKMs) (Hu et al. 2017a). SOL calpains have a conserved set of 5-6 N-terminal zinc fingers. Bioinformatic analysis suggests that these zinc fingers could bind to ubiquitin. In this study, we show that both the Aplysia and mouse SOL calpain (also known as Calpain 15) zinc fingers bind ubiquitinated proteins, and we confirm that Aplysia SOL binds poly- but not mono- or diubiquitin. No specific zinc finger is required for polyubiquitin binding. Neither polyubiquitin nor calcium was sufficient to induce purified Aplysia SOL calpain to autolyse or to cleave the atypical PKC to PKM in vitro. In Aplysia, over-expression of the atypical PKC in sensory neurons leads to an activity-dependent cleavage event and an increase in nuclear ubiquitin staining. Activity-dependent cleavage is partially blocked by a dominant negative SOL calpain, but not by a dominant negative classical calpain. The cleaved PKM was stabilized by the dominant negative classical calpain and destabilized by a dominant negative form of the PKM stabilizing protein KIdney/BRAin protein. These studies provide new insight into SOL calpain's function and regulation. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.


Subject(s)
Calpain/metabolism , Neurons/metabolism , Polyubiquitin/metabolism , Zinc Fingers/physiology , Animals , Aplysia , Cell Nucleus/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Neurons/ultrastructure , Protein Binding/genetics , Protein Kinase C-alpha/genetics , Protein Kinase C-alpha/metabolism , Statistics, Nonparametric , Transduction, Genetic , Red Fluorescent Protein
5.
Curr Biol ; 27(13): 1888-1899.e4, 2017 Jul 10.
Article in English | MEDLINE | ID: mdl-28648820

ABSTRACT

Generalization of fear responses to non-threatening stimuli is a feature of anxiety disorders. It has been challenging to target maladaptive generalized memories without affecting adaptive memories. Synapse-specific long-term plasticity underlying memory involves the targeting of plasticity-related proteins (PRPs) to activated synapses. If distinct tags and PRPs are used for different forms of plasticity, one could selectively remove distinct forms of memory. Using a stimulation paradigm in which associative long-term facilitation (LTF) occurs at one input and non-associative LTF at another input to the same postsynaptic neuron in an Aplysia sensorimotor preparation, we found that each form of LTF is reversed by inhibiting distinct isoforms of protein kinase M (PKM), putative PRPs, in the postsynaptic neuron. A dominant-negative (dn) atypical PKM selectively reversed associative LTF, while a dn classical PKM selectively reversed non-associative LTF. Although both PKMs are formed from calpain-mediated cleavage of protein kinase C (PKC) isoforms, each form of LTF is sensitive to a distinct dn calpain expressed in the postsynaptic neuron. Associative LTF is blocked by dn classical calpain, whereas non-associative LTF is blocked by dn small optic lobe (SOL) calpain. Interfering with a putative synaptic tag, the adaptor protein KIBRA, which protects the atypical PKM from degradation, selectively erases associative LTF. Thus, the activity of distinct PRPs and tags in a postsynaptic neuron contribute to the maintenance of different forms of synaptic plasticity at separate inputs, allowing for selective reversal of synaptic plasticity and providing a cellular basis for developing therapeutic strategies for selectively reversing maladaptive memories.


Subject(s)
Aplysia/physiology , Long-Term Potentiation/physiology , Memory/physiology , Neurons/physiology , Animals , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism
6.
Learn Mem ; 24(1): 1-13, 2017 01.
Article in English | MEDLINE | ID: mdl-27980071

ABSTRACT

Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that dominant-negative PKMs show isoform-specificity. A dominant-negative PKM from the classical PKC Apl I blocks activity-dependent intermediate-term facilitation (a-ITF) when expressed in the sensory neuron, while a dominant-negative PKM from the atypical PKC Apl III does not. Consistent with a specific role for PKM Apl I in activity-dependent facilitation, live imaging FRET-based cleavage assays reveal that activity leads to cleavage of the classical PKC Apl I, but not the atypical PKC Apl III in the sensory neuron varicosities of Aplysia In contrast, massed intermediate facilitation (m-ITF) induced by 10 min of 5HT is sufficient for cleavage of the atypical PKC Apl III in the motor neuron. Interestingly, both cleavage of PKC Apl I in the sensory neuron during a-ITF and cleavage of PKC Apl III in the motor neuron during m-ITF are inhibited by a dominant-negative form of a penta-EF hand containing classical calpain cloned from Aplysia Consistent with a role for PKMs in plasticity, this dominant-negative calpain also blocks both a-ITF when expressed in the sensory neuron and m-ITF when expressed in the motor neuron. This study broadens the role of PKMs in synaptic plasticity in two significant ways: (i) PKMs generated from multiple isoforms of PKC, including classical isoforms, maintain memory traces; (ii) PKMs play roles in the presynaptic neuron.


Subject(s)
Neuronal Plasticity/physiology , Presynaptic Terminals/physiology , Protein Kinase C/metabolism , Sensory Receptor Cells/cytology , Sensory Receptor Cells/metabolism , Animals , Aplysia , Benzophenanthridines/pharmacology , Calpain/pharmacology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer , Gene Expression Regulation/drug effects , Membrane Potentials/physiology , Microinjections , Motor Neurons/drug effects , Motor Neurons/physiology , Nervous System/cytology , Neuronal Plasticity/drug effects , Potassium Chloride/pharmacology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase C/chemistry , Protein Kinase C/drug effects , Protein Kinase C/genetics , Serotonin/pharmacology , Transduction, Genetic
7.
PLoS One ; 11(12): e0168411, 2016.
Article in English | MEDLINE | ID: mdl-28002451

ABSTRACT

Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT) underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF) receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF)-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs) or other molecules must also be involved in translocation of PKC Apl II.


Subject(s)
Aplysia/metabolism , Protein Kinase C/metabolism , Serotonin/pharmacology , Signal Transduction/drug effects , Adenylyl Cyclases/metabolism , Animals , Cells, Cultured , Coculture Techniques , ErbB Receptors/metabolism , Genistein/pharmacology , Isoenzymes/metabolism , Motor Neurons/cytology , Motor Neurons/drug effects , Motor Neurons/metabolism , Phosphorylation/drug effects , Phylogeny , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Receptors, Fibroblast Growth Factor/classification , Receptors, Fibroblast Growth Factor/metabolism , Sensory Receptor Cells/cytology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/metabolism , Sf9 Cells , Spodoptera , Synapses/metabolism
8.
J Neurosci ; 35(10): 4403-17, 2015 Mar 11.
Article in English | MEDLINE | ID: mdl-25762683

ABSTRACT

At the sensory-motor neuron synapse of Aplysia, either spaced or continuous (massed) exposure to serotonin (5-HT) induces a form of intermediate-term facilitation (ITF) that requires new protein synthesis but not gene transcription. However, spaced and massed ITF use distinct molecular mechanisms to maintain increased synaptic strength. Synapses activated by spaced applications of 5-HT generate an ITF that depends on persistent protein kinase A (PKA) activity, whereas an ITF produced by massed 5-HT depends on persistent protein kinase C (PKC) activity. In this study, we demonstrate that eukaryotic elongation factor 2 (eEF2), which catalyzes the GTP-dependent translocation of the ribosome during protein synthesis, acts as a biochemical sensor that is tuned to the pattern of neuronal stimulation. Specifically, we find that massed training leads to a PKC-dependent increase in phosphorylation of eEF2, whereas spaced training results in a PKA-dependent decrease in phosphorylation of eEF2. Importantly, by using either pharmacological or dominant-negative strategies to inhibit eEF2 kinase (eEF2K), we were able to block massed 5-HT-dependent increases in eEF2 phosphorylation and subsequent PKC-dependent ITF. In contrast, pharmacological inhibition of eEF2K during the longer period of time required for spaced training was sufficient to reduce eEF2 phosphorylation and induce ITF. Finally, we find that the massed 5-HT-dependent increase in synaptic strength requires translation elongation, but not translation initiation, whereas the spaced 5-HT-dependent increase in synaptic strength is partially dependent on translation initiation. Thus, bidirectional regulation of eEF2 is critical for decoding distinct activity patterns at synapses by activating distinct modes of translation regulation.


Subject(s)
Elongation Factor 2 Kinase/metabolism , Neuronal Plasticity/physiology , Neurons/metabolism , Synapses/physiology , Animals , Aplysia , Azides/metabolism , Cells, Cultured , Coculture Techniques , Dose-Response Relationship, Drug , Elongation Factor 2 Kinase/genetics , Enzyme Inhibitors/pharmacology , Ganglia, Invertebrate/cytology , Imidazoles/pharmacology , Insecta , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Membrane Potentials/drug effects , Membrane Potentials/genetics , Neuronal Plasticity/drug effects , Neurons/drug effects , Phosphorylation/drug effects , Phosphorylation/physiology , R-SNARE Proteins/genetics , R-SNARE Proteins/metabolism , Serotonin/pharmacology , Signal Transduction/drug effects , Spermine/analogs & derivatives , Spermine/metabolism , Synapses/drug effects
9.
Eur J Neurosci ; 41(3): 328-40, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25401305

ABSTRACT

Protein kinase Cs (PKCs) are activated by translocating from the cytoplasm to the membrane. We have previously shown that serotonin-mediated translocation of PKC to the plasma membrane in Aplysia sensory neurons was subject to desensitization, a decrease in the ability of serotonin to induce translocation after previous application of serotonin. In Aplysia, changes in the strength of the sensory-motor neuron synapse are important for behavioral sensitization and PKC regulates a number of important aspects of this form of synaptic plasticity. We have previously suggested that the desensitization of PKC translocation in Aplysia sensory neurons may partially explain the differences between spaced and massed training, as spaced applications of serotonin, a cellular analog of spaced training, cause greater desensitization of PKC translocation than one massed application of serotonin, a cellular analog of massed training. Our previous studies were performed in isolated sensory neurons. In the present study, we monitored translocation of fluorescently-tagged PKC to the plasma membrane in living sensory neurons that were co-cultured with motor neurons to allow for synapse formation. We show that desensitization now becomes similar during spaced and massed applications of serotonin. We had previously modeled the signaling pathways that govern desensitization in isolated sensory neurons. We now modify this mathematical model to account for the changes observed in desensitization dynamics following synapse formation. Our study shows that synapse formation leads to significant changes in the molecular signaling networks that underlie desensitization of PKC translocation.


Subject(s)
Cell Membrane/enzymology , Cytoplasm/enzymology , Protein Kinase C/metabolism , Synapses/physiology , Animals , Aplysia , Cell Membrane/drug effects , Coculture Techniques , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoplasm/drug effects , Models, Neurological , Motor Neurons/drug effects , Motor Neurons/physiology , Neuronal Plasticity , Protein Transport , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/physiology , Serotonin/metabolism , Synapses/drug effects
10.
J Neurosci ; 32(42): 14630-40, 2012 Oct 17.
Article in English | MEDLINE | ID: mdl-23077049

ABSTRACT

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.


Subject(s)
Aplysia/enzymology , Memory/physiology , Protein Kinase C/metabolism , Serotonin/physiology , Animals , Cell Line , Cells, Cultured , Isoenzymes/metabolism , Motor Neurons/enzymology
11.
J Neurochem ; 123(3): 360-72, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22913526

ABSTRACT

Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.


Subject(s)
Aplysia/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Sensory Receptor Cells/metabolism , Animals , Aplysia/enzymology , Marine Toxins , Mutagenesis/physiology , Oxazoles/pharmacology , Phosphatidic Acids/metabolism , Phosphorylation/drug effects , Phosphorylation/genetics , Protein Kinase C/genetics , Protein Kinase Inhibitors/pharmacology , Protein Processing, Post-Translational/genetics , Protein Structure, Tertiary/drug effects , Protein Structure, Tertiary/genetics , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/enzymology , Sf9 Cells
12.
Neural Plast ; 2012: 581291, 2012.
Article in English | MEDLINE | ID: mdl-22548194

ABSTRACT

Long-term memory formation is sensitive to the pattern of training sessions. Training distributed over time (spaced training) is superior at generating long-term memories than training presented with little or no rest interval (massed training). This spacing effect was observed in a range of organisms from invertebrates to humans. In the present paper, we discuss the evidence supporting cyclic-AMP response element-binding protein 2 (CREB), a transcription factor, as being an important molecule mediating long-term memory formation after spaced training. We also review the main upstream proteins that regulate CREB in different model organisms. Those include the eukaryotic translation initiation factor (eIF2α), protein phosphatase I (PP1), mitogen-activated protein kinase (MAPK), and the protein tyrosine phosphatase corkscrew. Finally, we discuss PKC activation and protein synthesis and degradation as mechanisms by which neurons decode the spacing intervals.


Subject(s)
Conditioning, Psychological/physiology , Cyclic AMP Response Element-Binding Protein/physiology , Memory/physiology , Animals , Humans , Mice , Mitogen-Activated Protein Kinases/physiology , Neurons/metabolism , Neurons/physiology , Protein Kinase C/physiology , Protein Phosphatase 1/physiology , Time Factors , Transcription Factors/physiology
13.
Adv Exp Med Biol ; 740: 663-83, 2012.
Article in English | MEDLINE | ID: mdl-22453964

ABSTRACT

More than two decades ago, the discovery of the first C2 domain in conventional Protein Kinase Cs (cPKCs) and of its role as a calcium-binding motif began to shed light on the activation mechanism of this family of Serine/Threonine kinases which are involved in several critical signal transduction pathways. In this chapter, we review the current knowledge of the structure and the function of the different C2 domains in PKCs. The C2 domain of cPKCs is a calcium sensor and its calcium-dependent binding to phospholipids is crucial for kinase activation. While the functional role of the cPKC C2 domain is better understood, phylogenetic analysis revealed that the novel C2 domain is more ancient and related to the C2 domain in the fungal PKC family, while the cPKC C2 domain is first associated with PKC in metazoans. The C2 domain of novel PKCs (nPKCs) does not contain a calcium-binding motif but still plays a critical role in nPKCs activation by regulating C1-C2 domain interactions and consequently C2 domain-mediated inhibition in both the nPKCs of the epsilon family and the nPKCs of the delta family. Moreover, the C2 domain of the nPKCs of the delta family was shown to recognize phosphotyrosines in a novel mode different from the ones observed for the Src Homology 2 (SH2) and the phosphotyrosine binding domains (PTB). By binding to phosphotyrosines, the C2 domain regulates the activation of this subclass of PKCs. The C2 domain was also shown to be involved in protein-protein interactions and binding to the receptor for activated C-kinase (RACKs) thus contributing to the subcellular localization of PKCs. In summary, the C2 domain is a critical player that can sense the activated signaling pathway in response to external stimuli to specifically regulate the different conventional and novel PKC isoforms.


Subject(s)
Protein Kinase C/physiology , Amino Acid Sequence , Animals , Calcium/metabolism , Evolution, Molecular , Humans , Molecular Sequence Data , Phospholipids/metabolism , Protein Kinase C/chemistry , Protein Structure, Tertiary , Signal Transduction
14.
J Neurophysiol ; 107(1): 292-305, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21994260

ABSTRACT

Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.


Subject(s)
Aplysia/physiology , Isoenzymes/metabolism , Neural Inhibition/physiology , Neurotransmitter Agents/metabolism , Protein Kinase C/metabolism , Sense Organs/physiology , Sensory Receptor Cells/physiology , Synaptic Transmission/physiology , Animals , Cells, Cultured , Enzyme Activation , Excitatory Postsynaptic Potentials/physiology , Pleura/innervation , Pleura/physiology
15.
Neurosci Lett ; 504(3): 306-10, 2011 Oct 31.
Article in English | MEDLINE | ID: mdl-21982802

ABSTRACT

Novel protein kinase Cs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. We have previously shown that the Aplysia novel PKC Apl II's C2 domain inhibits binding of diacylglycerol (DAG) to the C1 domain and that this inhibition is removed by phosphatidic acid (PA) binding to the C1b domain. Another model for C2 domain regulation of nPKCs suggests that the C2 domain binds to receptors for activated C kinase (RACKs) to assist in kinase translocation and activation. In the present study, we examined how a pharmacological peptide derived from RACK-binding site in the vertebrate novel PKCɛ regulates translocation of PKC Apl II from the cytosol to the plasma membrane. We found that a C2 domain-derived inhibitor peptide inhibited PKC Apl II translocation. This inhibition was removed by R273H mutation in the C1b domain and by phosphatidic acid, which can both remove C2-domain mediated inhibition suggesting that the peptide can regulate C1-C2 domain interactions.


Subject(s)
Calcium/metabolism , Isoenzymes/antagonists & inhibitors , Peptide Fragments/pharmacology , Protein Kinase C-epsilon/antagonists & inhibitors , Protein Kinase C/antagonists & inhibitors , Amino Acid Sequence , Animals , Aplysia , Binding Sites , Cell Line , Cell Membrane/metabolism , Consensus Sequence , Cytoplasm/metabolism , Diglycerides/metabolism , Drug Design , Isoenzymes/chemistry , Isoenzymes/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptide Fragments/chemical synthesis , Phosphatidic Acids/pharmacology , Point Mutation , Protein Conformation , Protein Interaction Mapping , Protein Kinase C/chemistry , Protein Kinase C/metabolism , Protein Structure, Tertiary , Protein Transport/drug effects , Rats , Sequence Alignment , Spodoptera
16.
J Vis Exp ; (50)2011 Apr 06.
Article in English | MEDLINE | ID: mdl-21505406

ABSTRACT

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, ßI, ßII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.


Subject(s)
Aplysia/enzymology , Microscopy, Confocal/methods , Neurons, Afferent/enzymology , Protein Kinase C/metabolism , Spodoptera/enzymology , Animals , Aplysia/chemistry , Aplysia/cytology , Cytological Techniques/methods , Fluorescent Dyes/chemistry , Isoenzymes , Neurons, Afferent/cytology , Protein Kinase C/analysis , Spodoptera/cytology
17.
PLoS Comput Biol ; 7(12): e1002324, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22219722

ABSTRACT

The sensory-motor neuron synapse of Aplysia is an excellent model system for investigating the biochemical changes underlying memory formation. In this system, training that is separated by rest periods (spaced training) leads to persistent changes in synaptic strength that depend on biochemical pathways that are different from those that occur when the training lacks rest periods (massed training). Recently, we have shown that in isolated sensory neurons, applications of serotonin, the neurotransmitter implicated in inducing these synaptic changes during memory formation, lead to desensitization of the PKC Apl II response, in a manner that depends on the method of application (spaced versus massed). Here, we develop a mathematical model of this response in order to gain insight into how neurons sense these different training protocols. The model was developed incrementally, and each component was experimentally validated, leading to two novel findings: First, the increased desensitization due to PKA-mediated heterologous desensitization is coupled to a faster recovery than the homologous desensitization that occurs in the absence of PKA activity. Second, the model suggests that increased spacing leads to greater desensitization due to the short half-life of a hypothetical protein, whose production prevents homologous desensitization. Thus, we predict that the effects of differential spacing are largely driven by the rates of production and degradation of proteins. This prediction suggests a powerful mechanism by which information about time is incorporated into neuronal processing.


Subject(s)
Aplysia/physiology , Memory , Neurons/pathology , Animals , Biochemistry/methods , Cyclic AMP-Dependent Protein Kinases/metabolism , Models, Biological , Models, Statistical , Models, Theoretical , Motor Neurons/physiology , Neuronal Plasticity/physiology , Plasmids/metabolism , Sensory Receptor Cells/physiology , Synaptic Transmission/physiology
18.
J Neurosci ; 29(33): 10281-6, 2009 Aug 19.
Article in English | MEDLINE | ID: mdl-19692602

ABSTRACT

Learning is highly regulated by the pattern of training. In Aplysia, an important organism for the development of cellular and molecular models of learning, spaced versus massed application of the same stimulus leads to different forms of memory. A critical molecular step underlying memory is the serotonin (5HT)-mediated activation of the novel PKC Apl II. Here, we demonstrate that activation of PKC Apl II is highly sensitive to the pattern of 5HT application. Spaced applications downregulate PKC translocation through PKA signaling, whereas massed applications lead to persistent translocation of PKC. Differential regulation of PKC translocation is mediated by competing feedback mechanisms that act through protein synthesis. These studies elucidate a fundamental molecular difference between spaced and massed training protocols.


Subject(s)
Aplysia/enzymology , Learning/physiology , Protein Kinase C/metabolism , Animals , Aplysia/drug effects , Cells, Cultured , Learning/drug effects , Memory/drug effects , Memory/physiology , Protein Biosynthesis/physiology , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Sensory Receptor Cells/drug effects , Sensory Receptor Cells/enzymology , Serotonin/pharmacology
19.
Mol Cell Biol ; 28(15): 4719-33, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18505819

ABSTRACT

In Aplysia californica, the serotonin-mediated translocation of protein kinase C (PKC) Apl II to neuronal membranes is important for synaptic plasticity. The orthologue of PKC Apl II, PKCepsilon, has been reported to require phosphatidic acid (PA) in conjunction with diacylglycerol (DAG) for translocation. We find that PKC Apl II can be synergistically translocated to membranes by the combination of DAG and PA. We identify a mutation in the C1b domain (arginine 273 to histidine; PKC Apl II-R273H) that removes the effects of exogenous PA. In Aplysia neurons, the inhibition of endogenous PA production by 1-butanol inhibited the physiological translocation of PKC Apl II by serotonin in the cell body and at the synapse but not the translocation of PKC Apl II-R273H. The translocation of PKC Apl II-R273H in the absence of PA was explained by two additional effects of this mutation: (i) the mutation removed C2 domain-mediated inhibition, and (ii) the mutation decreased the concentration of DAG required for PKC Apl II translocation. We present a model in which, under physiological conditions, PA is important to activate the novel PKC Apl II both by synergizing with DAG and removing C2 domain-mediated inhibition.


Subject(s)
Aplysia/enzymology , Isoenzymes/metabolism , Neurons, Afferent/enzymology , Phosphatidic Acids/metabolism , Protein Kinase C/metabolism , Amino Acid Sequence , Animals , Cell Line , Diglycerides/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Kinetics , Models, Biological , Molecular Sequence Data , Mutant Proteins/metabolism , Neurons, Afferent/drug effects , Phosphoinositide Phospholipase C/metabolism , Phospholipase D/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/chemistry , Protein Structure, Tertiary , Protein Transport/drug effects , Sequence Alignment , Serotonin/pharmacology
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